Application of aspergillus oryzae LCCC30141 in production of neutral protease and tobacco leaf fermentation
阅读说明：本技术 米曲霉lccc30141在生产中性蛋白酶和烟叶发酵中的应用 (Application of aspergillus oryzae LCCC30141 in production of neutral protease and tobacco leaf fermentation ) 是由 叶亚军 张庆华 田数 赵新海 陈晨 陈飞 张永岗 于宏男 刘月明 *** 李力群 于 2019-09-25 设计创作，主要内容包括：本发明提供了一种米曲霉(Aspergillus oryzae)LCCC30141在生产中性蛋白酶和烟叶发酵中的应用,米曲霉(Aspergillus oryzae)LCCC30141能够产生高酶活性的中性蛋白酶,利用米曲霉(Aspergillus oryzae)LCCC30141生产中性蛋白酶,为中性蛋白酶的生产来源提供了一个新的途径,可以实现中性蛋白酶的规模化生产；将具有产高酶活力的中性蛋白酶的米曲霉应用到烟叶发酵中,能够加速烟叶中蛋白质的分解,明显缩短烟叶的发酵周期,提升烟叶的品质。(The invention provides application of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 in production of neutral protease and tobacco fermentation, wherein the Aspergillus oryzae (Aspergillus oryzae) LCCC30141 can produce neutral protease with high enzyme activity, and the Aspergillus oryzae (Aspergillus oryzae) LCCC30141 is used for producing the neutral protease, so that a new way is provided for the production source of the neutral protease, and the scale production of the neutral protease can be realized; the aspergillus oryzae with the neutral protease capable of producing high enzyme activity is applied to the fermentation of the tobacco leaves, so that the decomposition of protein in the tobacco leaves can be accelerated, the fermentation period of the tobacco leaves is obviously shortened, and the quality of the tobacco leaves is improved.)
1. Use of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 in the production of a neutral protease.
2. A method for producing neutral protease by using Aspergillus oryzae (LCCC 30141), which comprises the step of subjecting Aspergillus oryzae LCCC30141 to fermentation culture to obtain a crude enzyme solution.
3. The method of claim 2, wherein the fermentation culture is a liquid fermentation culture or a solid fermentation culture.
4. The method according to claim 3, wherein the conditions of the liquid fermentation culture are: the inoculation amount is 105-107cfu/ml, the fermentation temperature is 20-40 ℃, the fermentation time is 2-4 d, and the obtained fermentation liquid is crude enzyme liquid;
preferably, the inoculum size is 106cfu/ml, the fermentation temperature is 30 ℃, and the fermentation time is 3 d;
preferably, the fermentation culture is ventilation culture, and the ventilation rate is 1: 0.4-0.6V/V.min;
preferably, the ventilation rate is 1: 0.5V/V.min;
preferably, the liquid fermentation medium used for the liquid fermentation culture contains: 30g/L of bran powder, 5g/L of yeast extract, 10g/L of corn steep liquor, 1g/L of ammonium sulfate, 2g/L of disodium hydrogen phosphate and 6.5-7.0 of pH.
5. The method according to claim 3, wherein the solid fermentation culture adopts a three-stage fermentation process, and specifically comprises the following steps:
(1) culturing Aspergillus oryzae (Aspergillus oryzae) LCCC30141 strain on slant PDA culture medium to obtain strain suspension;
(2) inoculating the strain suspension on a liquid strain culture medium for amplification culture to obtain a seed solution;
(3) mixing the seed liquid with the mixture at a ratio of 105-107cfu/g inoculated in solid cultureCulturing the medium at 20-40 ℃ for 2-4 days, adding physiological saline, leaching, and performing suction filtration to obtain a filtrate which is a crude enzyme solution;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar;
and/or the liquid seed culture medium contains: 30g/L of bran powder, 5g/L of yeast extract, 10g/L of corn steep liquor, 1g/L of ammonium sulfate, 2g/L of disodium hydrogen phosphate and 6.5-7.0 of pH;
and/or the solid medium contains: 40% of bran, 10% of soybean meal, 0.1% of disodium hydrogen phosphate, 0.05% of magnesium sulfate and 0.2% of ammonium sulfate.
6. The method of claim 2, further comprising the steps of adding ammonium sulfate to the crude enzyme solution for precipitation, dissolving the precipitate, dialyzing, centrifuging, and freeze-drying to obtain enzyme powder.
7. The method of claim 6, wherein the saturation of ammonium sulfate is 60-80%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-8000 r/min, and the time is 10-30 min.
8. Use of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 or the method of any of claims 2 to 7 for the fermentation of tobacco leaves.
9. A method for fermenting tobacco leaf is characterized in that Aspergillus oryzae (Aspergillus oryzae) LCCC30141 is inoculated in tobacco leaf for fermentation.
10. The method of fermenting tobacco leaves according to claim 9, wherein the amount of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 inoculated is 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
Technical Field
The invention relates to application of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 in production of neutral protease tobacco fermentation, and belongs to the technical field of microbial application.
Background
Neutral protease is also called serrapeptase, belongs to an endonuclease, and can be used for various proteolysis treatments. At a certain temperature and pH value, macromolecular protein in animals and plants can be hydrolyzed into products such as amino acid and the like. Aspergillus oryzae belongs to Aspergillus, Aspergillus flavus group Aspergillus oryzae is a main strain in China brewing industry, has the advantages of high protease activity, fast growth, strong adaptability, safety, no toxicity and the like, and is an excellent strain for producing neutral protease.
At present, most of the fermentation of tobacco leaves adopts natural fermentation and artificial fermentation. The natural fermentation is mainly carried out by means of the change of natural climate, and is mainly characterized in that the temperature rise in spring of one year is utilized to promote the activity of enzymes in the tobacco leaves during the storage period of the tobacco leaves, so that the tobacco leaves are subjected to a slow fermentation process to achieve the purpose of improving the quality of the tobacco leaves. The artificial fermentation is a method for accelerating the change of the tobacco leaves by utilizing artificial conditions (namely suitable temperature and humidity) suitable for the change of the internal quality of the tobacco leaves. However, both methods have the disadvantage that the fermentation progresses slowly.
Disclosure of Invention
The invention aims to provide application of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 in production of neutral protease and tobacco fermentation, wherein the Aspergillus oryzae (Aspergillus oryzae) LCCC30141 can produce neutral protease with high yield and is very suitable for production of neutral protease and tobacco fermentation.
In one aspect, the invention provides the use of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 in the production of neutral protease, wherein Aspergillus oryzae (Aspergillus oryzae) LCCC30141 can produce neutral protease with high enzymatic activity, thereby providing a new source for the production of neutral protease.
In another aspect, the present invention provides a method for producing a neutral protease using Aspergillus oryzae (Aspergillus oryzae) LCCC30141, comprising the step of subjecting Aspergillus oryzae (Aspergillus oryzae) LCCC30141 to fermentation culture to obtain a crude enzyme solution.
Preferably, the fermentation culture is a liquid fermentation culture or a solid fermentation culture.
Conditions of liquid fermentation culture: the inoculation amount is 105-107cfu/ml, the fermentation temperature is 20-40 ℃, the fermentation time is 2-4 d, and the obtained fermentation liquid is crude enzyme liquid;
preferably, the inoculum size is 106cfu/ml, the fermentation temperature is 30 ℃, and the fermentation time is 3 d;
preferably, the fermentation culture is ventilation culture, and the ventilation rate is 1: 0.4-0.6V/V.min;
preferably, the ventilation rate is 1: 0.5V/V.min;
preferably, the liquid fermentation medium used for the liquid fermentation culture contains: 30g/L of bran powder, 5g/L of yeast extract, 10g/L of corn steep liquor, 1g/L of ammonium sulfate, 2g/L of disodium hydrogen phosphate and 6.5-7.0 of pH.
Preferably, the solid fermentation culture adopts a three-stage fermentation process, and specifically comprises the following steps:
(1) culturing Aspergillus oryzae LCCC30141 strain on slant PDA culture medium to obtain strain suspension;
(2) inoculating the strain suspension on a liquid strain culture medium for amplification culture to obtain a seed solution;
(3) mixing the seed liquid with the mixture at a ratio of 105-107Inoculating cfu/g into a solid culture medium, culturing at 20-40 ℃ for 2-4 days, adding physiological saline, leaching, and performing suction filtration to obtain a filtrate which is a crude enzyme solution;
preferably, the PDA medium contains: 200g/L of potato, 20g/L of glucose and 15g/L of agar;
and/or the liquid seed culture medium contains: 30g/L of bran powder, 5g/L of yeast extract, 10g/L of corn steep liquor, 1g/L of ammonium sulfate, 2g/L of disodium hydrogen phosphate and 6.5-7.0 of pH;
and/or the solid medium contains: 40% of bran, 10% of soybean meal, 0.1% of disodium hydrogen phosphate, 0.05% of magnesium sulfate and 0.2% of ammonium sulfate;
further, the method also comprises the steps of adding ammonium sulfate into the crude enzyme liquid for precipitation, dialyzing, centrifuging and freeze-drying the dissolved precipitate to obtain enzyme powder.
Preferably, the saturation degree of the ammonium sulfate is 60-80%;
preferably, the centrifugation temperature is 0-4 ℃, the rotation speed is 5000-8000 r/min, and the time is 10-30 min.
In another aspect, the invention also provides the use of Aspergillus oryzae (Aspergillus oryzae) LCCC30141 or the method in tobacco leaf fermentation.
In another aspect, the invention also provides a tobacco leaf fermentation method, wherein Aspergillus oryzae (Aspergillus oryzae) LCCC30141 is inoculated in tobacco leaves for fermentation.
Preferably, Aspergillus oryzae (Aspergillus oryzae) LCCC30141 is inoculated in an amount of 105～107cfu/g, the fermentation temperature is 20-40 ℃, the humidity is 40-70%, and the time is 5-10 d.
The invention has the beneficial effects that:
the invention discloses Aspergillus oryzae (Aspergillus oryzae) LCCC30141 capable of producing neutral protease with high enzyme activity, which provides a new way for the production source of the neutral protease and can realize the scale production of the neutral protease; the Aspergillus niger with the neutral protease with high enzyme activity is applied to the tobacco leaf fermentation, so that the decomposition of protein in the tobacco leaf can be accelerated, the fermentation period of the tobacco leaf is obviously shortened, and the quality of the tobacco leaf is improved.
Detailed Description
The present invention is described in detail with reference to specific examples, which are provided to facilitate the understanding of the technical solutions of the present invention by those skilled in the art, and the implementation or use of the present invention is not limited by the description of the present invention.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, if not specified. The Aspergillus oryzae (Aspergillus oryzae) strain used in the invention is purchased from Liaoning province microorganism strain preservation center, and the serial numbers are as follows: LCCC 30141.